We have made significant progress in developing a sensitive and reproducible method for probing the global transcriptome of C. burnetii. Transcription microarray analysis of obligate intracellular bacteria is technically challenging due to the vast abundance of host cell RNA, especially at early time points post infection. To circumvent this problem, we optimized several steps of the microarray procedure which included 1) adding a mechanical disruption step following chemical lysis of host cells to release RNA from resistant C. burnetii, 2) using a MicroEnrich kit to remove polyadenylated eucaryotic RNAs from RNA samples 3) employing of a MessageAmp II-Bacteria kit to amplify C. burnetii mRNA and, 4) increasing the microarray hybridization time to increase signal intensity. Indeed, data derived from the first application of this protocol was instrumental in development of our axenic growth medium ACCM (described in project ZOI AI000931-06 Cellular and Developmental Biology of Coxiella burnetii). By comparing the transcriptomes of C. burnetii growing in Vero cells and metabolizing in CCM medium (a precursor of ACCM), we discovered a cluster of ribosomal genes that were dramatically down-regulated by C. burnetii in CCM, suggesting insufficient levels of amino acids in this medium. Increased levels of amino acids in ACCM, combined with a microaerophilic environment, then led to axenic growth of C. burnetii. With microarray technology in hand, we are now poised to 1) define the developmental gene regulation that occurs during small cell to large cell variant morphological differentiation 2) examine transcriptional responses to stresses likely encountered in the phagolysosome (e.g., oxidative stress), and 3) compare the transcriptome of ACCM-cultivated and host cell-propagated organisms to identify genes that are upregulated during infection. [unreadable] [unreadable] Genetically distinct isolates of C. burnetii display different phenotypes with respect to in vitro infectivity/cytopathology and pathogenicity for laboratory animals. Moreover, correlations between C. burnetii genomic groups and human disease presentation (acute versus chronic) have been described, suggesting isolates have distinct virulence characteristics. To provide a more complete understanding of C. burnetii genetic diversity, evolution, and pathogenic potential, we have deciphered the whole genome sequences of the K (Q154) and G (Q212) human chronic endocarditis isolates and the naturally attenuated Dugway (5J108-111) rodent isolate. As previously described for the Dugway isolate, K and G isolates were significantly reduced in virulence for Balb/C mice when compared to the Nine Mile (NM) (RSA493) reference isolate. Cross-genome comparisons that included previously-sequenced NM revealed both novel gene content and disparate collections of pseudogenes that may contribute to isolate virulence and other phenotypes. While C. burnetii genomes are highly syntenous, recombination between abundant insertion sequence (IS) elements has resulted in genome plasticity manifested as chromosomal rearrangement of syntenic blocks and DNA insertions/deletions. The numerous IS elements, genomic rearrangements, and pseudogenes of C. burnetii isolates is consistent with genome structures of other bacterial pathogens that have recently emerged from non-pathogens with expanded niches. The observation that the severely attenuated Dugway isolate has the largest genome with the fewest pseudogenes and IS elements suggests this isolate lineage is at an earlier stage of pathoadaptation than the NM, K, and G lineages.[unreadable] [unreadable] We have successfully genetically transformed C. burnetii using a plasmid containing a Himar1 transposon (Tn) encoding chloramphenicol resistance and mCherry red fluorescent protein under control of a constitutive C. burnetii Hsp20 (CBU1169) promoter, and a ColE1 origin of replication. Chloramphenicol is not used in the clinical treatment of human Q fever and therefore its specific resistance gene is acceptable for use in genetic transformation studies. C. burnetii was co-electroporated with the Tn-containing plasmid and a plasmid encoding the Himar1 transposase variant C9 (also under the control of p1169). Transformants replicated in Vero cells in the presence of an inhibitory concentration of chloramphenicol (5 &#956;g/ml) and expressed mCherry. Rescue cloning of the ColE1 origin of replication and DNA sequencing revealed 35 different (Tn) insertion sites scattered throughout the C. burnetii genome. Thirty insertions were within coding regions while 5 were intergenic. A clone from the transformant mixture was isolated using our new micromanipulation cloning method and shown to harbor a Tn insertion within the essential cell division gene ftsZ that disrupts the reading frame at aa 368 of the 393 aa protein. Characterization of FtsZ::Tn mutant revealed a generation time during exponential phase of 19.8 h, almost twice as long as wild type C. burnetii (11.7 h). Thus, the Himar1 transposon system is a robust technique for creating genetic mutations in C. burnetii. [unreadable] [unreadable] Lipopolysaccharide is the only defined virulence factor of C. burnetii. Virulent phase I organisms, producing full-length LPS, convert to avirulent phase II organisms, synthesizing severely truncated LPS, upon repeat in vitro passages. The genetic lesion(s) accounting for the deep rough phenotype of phase II isolates is unknown. To this end, we generated phase II clones of the high passage Australian and California strains, using our new micromanipulation cloning procedure, and hybridized their genomic DNAs to a high-density microarray that contains probe sets encompassing all full-length open reading frames of the Nine Mile phase I strain. These arrays are specifically designed to detect indels (insertions/deletions). A common indel was found within a gene involved in heptose biosynthesis that we believe accounts for phase conversion. We are currently testing this hypothesis by expressing mutant and wild type versions of the gene in Salmonella enterica and scoring for altered LPS biosynthesis. [unreadable] [unreadable] There is a need for sensitive and specific serodiagnostic tests utilizing recombinant C. burnetii protein(s). To pursue this goal, we developed a C. burnetii protein microarray to comprehensively identify immunodominant antigens recognized by antibody in the context of human C. burnetii infection or vaccination. Transcriptionally active PCR (TAP) products corresponding to 1988 C. burnetii open reading frames (ORFs) were generated. Full-length proteins were successfully synthesized from 75% of the ORFs by using an Escherichia coli-based cell-free in vitro transcription and translation system (IVTT). Nitrocellulose microarrays were spotted with crude IVTT lysates and probed with sera from acute Q fever patients and individuals vaccinated with Q-Vax. Immune sera strongly reacted with approximately 50 C. burnetii proteins including previously identified immunogens, an ankyrin repeat-domain containing protein, and multiple hypothetical proteins. Recombinant protein corresponding to selected array-reactive antigens was generated and immunoreactivity confirmed by ELISA. This sensitive and high throughput method for identifying immunoreactive C. burnetii proteins will aid development of Q fever serodiagnostic tests based on recombinant antigen. Moreover, testing of microarray-identified antigens for T-cell antigenicity may identify proteins with efficacy as subunit vaccines against Q fever. As detailed below in our scientific advance , such testing does not require purified recombinant protein as IVTT-produced antigen coupled to latex beads by an affinity tag effectively stimulates T-cells in proliferation assays.